Modular PKS enzymes are large, multi-subunit enzyme complexes that perform the biosynthesis of polyketide secondary metabolites. See O'Hagan, D., 1991 (a full citation of any reference referred to herein by last name of first author and year of publication is located at the end of this section). Examples of polyketides made by modular PKS enzymes include the antibiotic erythromycin, the immunosuppressant FK506, and the antitumor compound epothilone. See also PCT patent publication No. 93/13663 (erythromycin); U.S. Pat. No. 6,303,342 B1 (epothilone); U.S. Pat. No. 6,251,636 B1 (oleandolide); PCT publication WO 01/27284 A2 (megalomicin); U.S. Pat. No. 5,098,837 (tylosin); U.S. Pat. No. 5,272,474 (avermectin); U.S. Pat. No. 5,744,350 (triol polyketide); and European patent publication No. 791,656, now U.S. Pat. No. 5,945,320 (platenolide), each of which is incorporated herein by reference.
PCT publication WO 01/27284 A2 referenced above discloses the desosamine biosynthesis gene megCII encoding a 3,4-isomerase and glycosylyltransferase gene megCIII; the mycarose biosynthesis genes megBII (megBII-2) and megBIV encoding a 2,3-reductase and 4-ketoreductase respectively, and the mycarose glycosyltransferase gene megBV; the megosamine biosynthesis genes megDII, megDIII, megDIV, megDV, and megDVI, and the megosanine glycosyltransferase gene megDI. That publication made partial disclosures of megBVI (megT) and megF. The megBVI gene, which has dual function in mycarose and megosamine biosynthesis as a 2,3-dehydratase, was only partially disclosed (less than 10% of the nucleotide sequence) and was named megT. The megF genes sequence was disclosed in part (47%).
A large interest in PKS enzymes arises from the ability to manipulate the specificity or sequence of reactions catalyzed by PKSs to produce novel useful compounds. See U.S. Pat. No. 5,962,290 and McDaniel, R., et al., 2000, and Weissman, K. J et al. 2001. A number of plasmid-based heterologous expression systems have been developed for the engineering and expression of PKSs, including multiple-plasmid systems for combinatorial biosynthesis. See McDaniel, et al., 1993, Xue, et al., 1999, and Ziermann, et al., 2000, and U.S. Pat. Nos. 6,033,883 and 6,177,262; and PCT publication Nos. 00/63361 and 00/24907, each of which is incorporated herein by reference. Polyketides are often modified by P450 enzymes that hydroxylate the polyketide and by glycosyl transferase enzymes that glycosylate the polypeptide. Using recombinant technology, see PCT Pub. No. 98/49315, incorporated herein by reference, one can also hydroxylate and or glycosylate polyketides. Such technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
The class of polyketides includes the megalomicins, which are 6-O-glycosides of erythromycin C with acetyl or propionyl groups esterified to the 3′″ or 4′″ hydroxyls of the mycarose sugar. They were reported in 1969 as antibacterial agents produced by Micromonospora megalomicea sp. n. (Weinstein et al., 1969). The deoxyamino sugar at C-6 was named “megosamine” (Nakagawa et al., 1984). Therapeutic interest in megalomicin arose from several observed biological activities, including anti-bacterial activity, effects on protein trafficking in eukaryotic cells, inhibition of vesicular transport between the medial and trans Golgi, resulting in undersialylation of proteins, inhibition of the ATP-dependent acidification of lysosomes, anomalous glycosylation of viral proteins, antiviral activity against herpes, and as potent antiparasitic agents. Megalomicins are effective against Plasmodium falciparum, Trypanosoma sp. and Leishmania donovani (Bonay et al., 1998). As erythromycin does not have antiparasitic activity, the antiparasitic action of megalomicin is most probably related to the presence of the megosamine deoxyamino sugar at C-6.
The aglycone backbone of both megalomicin and erythromycin is the complex polyketide 6-deoxyerythronolide B (6-dEB), produced from the successive condensations of a propionyl-CoA starter unit and 6 methylmalonyl-CoA extender units (FIG. 2). Complex polyketides are assembled by modular polyketide synthases (PKSs), which are composed of multifunctional polypeptides that contain the activities (as enzymatic domains) for the condensation and subsequent reductions required to produce the polyketide chain (Katz, 1997; Cane et al., 1998).
The biosynthetic pathway of megalomicin is shown in FIG. 2. Both the megalomicin and erythromycin pathways are identical through the formation of erythromycin C, the penultimate intermediate of erythromycin A and megalomicin A. The megalomicin biosynthetic gene cluster has, in addition to the genes for the synthesis and attachment of the mycarose and desosamine sugars, a set of genes for synthesis and attachment of the unique deoxysugar L-megosamine. Making glycosylated and or/hydroxylated derivatives of aglycones through genetic engineering would be possible if one could transfer one or more of the megalomicin sugar biosynthesis and glycosyl-transferase, and P450 monooxygenase genes to another host. There exists a need for methods and materials to modify polyketides by P450 modification and/or the addition of sugar moieties to create active compounds in heterologous or native hosts. The present invention provides methods and compositions to meet those and other needs.
The following articles provide background information relating to the invention and are incorporated herein by reference.    Alarcon, B., et al. (1984), Antiviral Res 4: 231-243.    Alarcon, B., et al (1988), FEBS Lett 231:207-211.    Altschul, S. F., et al. (1990), J Mol Biol 215: 403-410.    Andersen, J. F., et al. (1992), J Bacteriol 174: 725-735.    Arisawa, A., et al. (1993), Biosci Biotechnol Biochem 57: 2020-2025.    Arisawa, A., et al. (1994), Appl Environ Microbiol 60:2657-2660.    Bierman, M., et al. (1992), Gene 118: 43-49.    Bisang, C., et al. (1999), Nature 401: 502-505.    Bonay, P., et al. (1996), J Biol Chem 271: 3719-3726.    Bonay, P., et al. (1997), J Cell Sci 110:1839-1849 (1997).    Bonay, P., et al. (1998), Antimicrob Agents Chemother 42: 2668-2673.    Brünker, P., et al. (1998), Microbiology 144: 2441-2448.    Butler, A. R., et al. (1999), Chem Biol 6: 287-292.    Cane, D. E., et al. (1998), Science 282: 63-68.    Cortés, J., et al. (1990), Nature 348:176-178.    Dhillon, N., et al. (1989), Mol Microbiol 3:1405-1414.    Donadio, S., and Katz, L. (1992), Gene 111: 51-60.    Donadio, S., et al. (1993), Gene 126: 147-151.    Donadio, S., et al. (1991), Science 252: 675-679.    Epp, J. K., et al. (1989), Gene 85: 293-301.    Gaisser, S., et al. (1997), Mol Gen Genet 256: 239-251.    Gokhale, R. S., et al. (1999), Science 284: 482-485.    Gu, H., et al. (1996), Clin J Biotechnol 12:147-152.    Hara, O., et al. (1992), J Bacteriol 174:5141-5144.    Haydock, S. F., et al. (1991), Mol Gen Genet 230: 120-128.    Hopwood, D. A., et al. (1985) Genetic Manipulation of Streptomyces: A Laboratory Manual. Norwich, UK: The John Innes Foundation.    Kakavas, S. J., Katz, L., and Stassi, D. (1997), J Bacteriol 79: 7515-7522.    Kao, C. M., et al. (1994a), J Am Chem Soc 116: 11612-11613.    Kao, C. M., et al. (1994b), Science 265: 509-512.    Katz, L. (1997), Chem Rev 97: 2557-2576.    Kuhstoss, S., et al. (1996), Gene 183:231-236.    McDaniel, R., et al. (1993), Science 262:1546-1557.    McDaniel, R., et al. (1999), Proc Natl Acad Sci USA 96:1846-1851.    McDaniel, R., et al. (2000), Adv Bio Eng, 73: 31-52.    Nakagawa, A., et al. (1984) Structure and stereochemistry of macrolides. In Macrolide Antibiotics. Omura, S. (ed.). New York: Academic Press, pp. 37-84.    O'Hagan, D., et al. (1991) The polyketide metabolites. Ellis Horwood, Chichester, UK.    Olano, C., et al. (1999), Chem Biol 6: 845-855.    Pereda, A., et al. (1997), Gene 193: 65-71.    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.    Schwecke, T., et al. (1995), Proc Natl Acad Sci USA 92: 7839-7843.    Shah, S., et al. (2000), J Antibiotics 53: 502-508.    Stassi, D., et al. (1993), J Bacterial 175:182-189.    Summers, R. G., et al. (1997), Microbiology 143: 3251-3262.    Tang, L, et al. (1999), Chem Biol 6: 553-558.    Tang, L., et al. (2000), Chem Biol 7: 77-84.    van Wageningen, A., et al. (1998), Chem Biol 3:155-162.    Volchegursky, Y., et al. (2000), Mol Microbiology 37(4), 752-762.    Weber, J. M., et al. (1990), J Bacteriol 172: 2372-2383.    Weber, J. M., et al. (1991), Science 252: 114-117.    Weinstein, M. J., et al. (1969), J Antibiot 22: 253-258.    Weissman, K. J., et al. (2001), In H. A. Kirst et al. (ed.), Enzyme technologies for pharmaceutical and biotechnological applications, p. 427-470. Marcel Dekker, Inc. New York.    Xue, O., et al. (1999), Proc Natl Acad Sci USA 96:11740-11745.    Xue, Y., et al. (1998), Proc Natl Acad Sci USA 95: 12111-12116.    Zhao, L., et al. (1998), J Am Chem Soc 120: 10256-10257.    Ziermann, R., et al. (1999), Biotechniques 26: 106-110.    Ziermann, R., et al. (2000), J Ind Microbial Biotech 24: 46-50.